Notice: NOT-OD-09-058, NIH Announces the Availability of Recovery Act Funds for Competitive Applications SUMMARY STATEMENT "Group Theoretic Methods in Protein Structure Determination" The emphasis of this supplemental application is to analyze the statistics of ensembles of conformations of flexible proteins. That is, whereas the original grant focuses on new algorithms for determining protein structures from a variety of experimental modalities, the goal of this supplemental application is to quantify the variability of conformations represented in structural data. One of the most obvious examples of this is in NMR, where ensembles of conformations are often reported rather than an individual structure. The following specific aims to be addressed in this highly focused nine-month effort add to the original three specific aims: Specific Aim 4: Efficient Generation of Ensemble Statistics of Flexible Loop Conformations. In this specific aim, new methods from the field of robotic manipulator kinematics based on the concept of convolution and covariance propagation on the group of rigid-body motions will be used to generate ensemble statistics of candidate loop conformations without explicitly sampling conformational space. By circumventing the exponential price associated with traditional sampling techniques, we anticipate making a qualitative difference in the way protein-loop conformations are analyzed, not just a quantitative difference in particular computations. As a concrete test case, we will apply this methodology to the structure determination of human Ube2g2. Specific Aim 5: Evaluation of the Biasing Effects of Loops in the Assembly of Secondary Structures. In this specific aim we will study the correlation between loop length/composition and the special geometric characteristics of the resulting secondary structure interactions. We will begin by considering the helix-helix interaction in the helix-loop-helix motif and the relationship between helix interaction parameters and the parameters defining the loop. In other words, we will examine geometric characteristics such as crossing angle and where along the helix lengths the helices contact each other that depends on loop parameters. This is a purely in- silico objective, which will involve mining data from the Protein Data Bank. Specific Aim 6: Characterizing the Tertiary Ensemble. We will evaluate the following hypothesis: Protein tertiary structures do not always exist as single conformations, and enumeration of members of the 'tertiary ensemble'will better match experimental data than models in which a single conformation is assumed. We will test this hypothesis by validating tertiary ensembles against NMR residual dipolar couplings. In particular, we propose to utilize Neisseria meningitidis Heme Oxygenase (nmHO) as a test case of our ability to generate tertiary ensembles which maintain, or even improve, agreement with NMR data such as RDCs. Since the original grant is currently in no-cost extension, and almost all of the funds have been spent, this competitive renewal will provide an avenue for the continued funding of PI/co-PI salaries, a postdoc, and two students for a nine-month period. This, together with the proposed budget for laboratory supplies, constitutes the proposed request. PUBLIC HEALTH RELEVANCE: The emphasis of this supplemental application is to analyze the statistics of ensembles of conformations of flexible proteins. That is, whereas the original grant focuses on new algorithms for determining protein structures from a variety of experimental modalities, the goal of this supplemental application is to quantify the variability of conformations represented in structural data.